Our overall goal is to integrate sequence data with atomic-resolution 3D structures of the ribosomal RNAs (5S, 16S-like, and 23S-like rRNAs), so as to generate accurate structural alignments that make full use of both the sequence and 3D data. The end result will be the development of new on-line software tools for automating the process of adding new sequences to the database of structurally aligned sequences and for evaluating those alignments for novel motifs. The resulting alignments will improve the utility of the ribosomal RNAs for phylogenetic studies at all levels and will provide a basis for 3D modeling of the ribosomes of organisms of emerging medical interest where crystal structures are not available. This proposal aims to accomplish specific subgoals required for achieving the overall goal, building on the accomplishments of the previous grant period, which include the creation of a comprehensive yet easily understood classification and compilation of non-Watson-Crick basepairs (made available on-line) and a systematic approach to annotating, classifying, and aligning RNA motifs, defined as ordered arrays of non-Watson-Crick basepairs. The subgoals include: 1) Annotating, classifying, and cataloguing all RNA motifs in the atomic-resolution structures of ribosomal subunits. 2) Structural alignment of the archaeal (H. marismortui) and bacterial (D. radiodurans) 5S and 23S sequences (for which we have atomic-resolution 3D structures) to identify conserved features, motif swaps, and points of variation. 3) Comparison of Isostericity Matrices (I.M.) derived from structural analysis with sequence variations in the ribosomal RNAs to iteratively refine the I.M.'s and identify regions in the alignments which require reworking 4) Identification of "sequence signatures" of recurrent motifs and Molecular Dynamics simulation of commonly occurring sequence variations to refine 3D structural models deduced by isosteric modeling. 5) Thermodynamic studies of RNA-RNA interactions mediated by recurrent motifs. Motifs will be modularly inserted in tecto-RNA molecules we have computer designed to study RNA-RNA self-assembly. UV melting experiments, Isothermal Titration Calorimetry and CD spectroscopy will be employed to characterize interactions as functions of temperature and magnesium concentration. [unreadable] [unreadable]